NMDA receptors play important and diverse roles in CNS function, ranging from the regulation of synaptic plasticity and neuronal growth and survival to the initiation of cell death. It is generally agreed that mitochondrial calcium (Ca2+) overload and subsequent dysfunction, due to excessive Ca2+ entry through glutamate-overactivated NMDA receptors (NMDARs), are crucial early events in excitotoxic injury. However, there are diverse and seemingly conflicting viewpoints concerning the mechanisms underlying NMDAR susceptibility to overactivation. [unreadable] [unreadable] The overall goals of this project are: 1) to establish the cellular mechanisms by which mitochondria mediate glutamate-induced, calcium-dependent toxicity; and 2) to determine the mechanism of mitochondrial injury per se and devise ways to prevent it. This project is generally informed by the unifying hypothesis that calcium-induced mitochondrial dysfunction is the indispensable, central event in excitotoxicity, so that the effects of other known factors can be explained by their common, convergent impact on calcium load-dependent mitochondrial injury.[unreadable] [unreadable] Aim #1: To determine whether mitochondrial calcium overload can account for the role of NMDAR location and subunit composition in excitotoxic injury.[unreadable] Excitotoxicity depends on the activation of specific routes of Ca2+ entry. For example, NMDAR location appears to play an important role in specifying the induction of survival vs. death pathways, with extrasynaptic NMDARs being linked to excitotoxic death, while synaptic NMDARs promote cell survival. Alternatively, it has been suggested that excitotoxicity is triggered by the selective activation of those NMDARs containing the NR2B subunit. This projects working hypothesis proposes, and preliminary data support, the idea that the effects of factors such as location and composition can be accounted for by a common, convergent impact on calcium-induced mitochondrial injury. Thus, exposure of cultured rat hippocampal neurons to 100 uM NMDA normally leads to massive calcium accumulation and cell death. In cells expressing comparable levels of NR2A- and NR2B-containing NMDARs, inhibition of both receptor subtypes was necessary to reduce NMDA-evoked calcium elevations, preserve mitochondrial structure and function, and achieve satisfactory neuroprotection. This indicates that that toxic calcium loading can be mediated by receptors of either subtype, and therefore that subunit composition alone is not sufficient to specify signal coupling. Selective activation of extrasynaptic NMDARs resulted in calcium loads and toxicity that were comparable to global activation, while synaptic stimulation produced only low-amplitude calcium spikes that were pro-survival. These results imply that extrasynaptic NMDARs represent the major mediator of excitotoxic stimuli, precisely because they are the dominant route of calcium entry. The results support the hypothesis that excessive calcium loading and mitochondrial dysfunction are common, obligatory steps along the pathway to excitotoxic injury.[unreadable] [unreadable] Aim #2: To test the hypothesis that calcium overloadinduced mitochondrial damage is greater in CA1 neurons than in CA3 neurons, thereby explaining why CA1 neurons are more vulnerable to ischemic injury. To determine whether ischemic preconditioning works by reducing mitochondrial Ca overload in CA1 neurons. [unreadable] Slice cultures of hippocampus are a good model for studying neuronal tolerance, since pyramidal neurons of the CA1 region are quite sensitive to excitotoxic stimuli, while neurons in the CA3 region show a high level of endogenous neuroprotection. Present data indicate that NMDA exposure selectively induces large calcium elevations in CA1 neurons, but not in CA3 neurons. Consistent with the general principle that mitochondrial damage is a key event in excitotoxic vulnerability, mitochondrial calcium overload and damage were also much more severe in CA1. The NMDA antagonist MK-801 prevented CA1 calcium elevation and was neuroprotective. Considering results described in Aim #1, it is of interest to examine the role of route specificity in CA1 relative to CA3 to determine if mitochondrial calcium overload is generally responsible for the excitotoxic vulnerability of CA1 neurons.[unreadable] [unreadable] Aim #3: To determine if mitochondrial swelling during excitotoxic overstimulation is caused by calcium-induced opening of the mitochondrial permeability transition (MPT) pore.[unreadable] Calcium loading and oxidative stress are observed clinically during ischemic episodes. Although this suggests a role for MPT in excitotoxic injury, this idea remains controversial. In isolated mitochondria MPT is associated with the loss of the mitochondrial membrane potential (MMP), permeabilization of the inner mitochondrial membrane, swelling of the matrix, and outer membrane rupture, followed by release of apoptogenic proteins. These are all hallmarks of mitochondrial injury leading to excitotoxic death. Like mitochondria in intact neurons, isolated brain mitochondria are capable of accumulating large amounts of calcium when exposed to submicromolar concentrations of this ion. Accumulated calcium is stored in the matrix as phosphorus-rich precipitates, the chemical composition of which is largely unknown. Also unknown is the fate of these precipitates when the inner mitochondrial membrane is breached. This study aims to determine how the amount and rate of mitochondrial calcium uptake relate to MPT, precipitate composition and precipitate retention. Various inhibitors of MPT altered the Ca/P ratio of formed precipitates, indicating that precipitate chemical form and solubility vary with the conditions of loading. Following MPT the release of accumulated Ca2+ is rapid but incomplete; significant residual calcium is retained in damaged mitochondria for prolonged periods. Since mitochondrial Ca2+ transport significantly influences cell signaling, it seems desirable to examine whether prolonging Ca2+ release will have a significant impact during post-stimulus recovery periods.[unreadable] [unreadable] Aim #4: To develop new technology, based on energy filtering transmission electron microscopy (EFTEM), for the quantitative mapping of intracellular calcium distributions at the single organelle level. To apply this technology to elucidating spatio-temporal calcium dynamics in a model neuron.[unreadable] Because calcium plays such a major role in physiological and pathophysiological processes, quantitative mapping of Ca distributions in the analytical electron microscopy at the level of cellular organelles would be very helpful for elucidating mechanisms of calcium regulation and calcium-dependent dysfunction. However, since physiological concentrations of calcium are very low this task is challenging. In this project we propose mapping calcium using EFTEM. With EFTEM one can obtain megapixel images from cells with relatively short (<1 min) exposures, which greatly improves throughput and efficiency relative to alternative approaches. The main hurdle to producing reliable results from biological specimens is that thickness-dependent systematic errors in subtracting the spectral background from the weak, calcium-specific core edge signal must be eliminated. Present results show that by modeling the behavior of the spectrum background as a function of specimen thickness and inelastic mean free path, we can correct for plural scattering and detect calcium in neuronal preparations at concentrations below 10 mmol/kg dry weight, which is in the high physiological range. Results also suggest that is will be quite feasible to further reduce these detection limits. The first proposed biological application of this methodology will be directed toward understanding calcium dynamics during recovery from stimulated calcium entry in frog sympathetic neurons, a model that we have studied in in the past.